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Nitrosopyridine probe to detect polyketide natural products with conjugated alkenes: Discovery of novodaryamide and nocarditriene Gabriel Castro-Falcón, Natalie Millán Aguiñaga, Catherine Roullier, Paul R Jensen, and Chambers C. Hughes ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.8b00598 • Publication Date (Web): 01 Oct 2018 Downloaded from http://pubs.acs.org on October 5, 2018

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ACS Chemical Biology

Nitrosopyridine probe to detect polyketide natural products with conjugated alkenes: Discovery of novodaryamide and nocarditriene

Gabriel Castro-Falcón,1 Natalie Millán-Aguiñaga,1 Catherine Roullier,2 Paul R. Jensen,1 Chambers C. Hughes1*

1

Center for Marine Biotechnology and Biomedicine, Scripps Institution of

Oceanography, University of California, San Diego, La Jolla, California, USA

2

UNIV Nantes, Université Bretagne Loire, Nantes 44035 cedex 1, France

*e-mail: [email protected]

1 ACS Paragon Plus Environment

ACS Chemical Biology 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract An optimized nitroso-based probe that facilitates the discovery of conjugated alkene-containing natural products in unprocessed extracts was developed. It chemoselectively reacts with conjugated olefins via a nitroso-Diels Alder cyclization to yield derivatives with a distinct chromophore and an isotopically-unique bromine atom that can be rapidly identified using liquid chromatography/mass spectrometry and a bioinformatics tool called MeHaloCoA (Marine Halogenated Compound Analysis). The probe is ideally employed when genome-mining techniques identify strains containing one or more polyketide gene clusters with two or more repeating KS-AT-DH-KR-ACP domain sequences, which are required for the biosynthesis of conjugated alkenes. Comparing the reactivity and spectral properties of five brominated arylnitroso reagents with model compounds spiramycin, bufalin, rapamycin, and rifampicin led to the identification of 5-bromo-2-nitrosopyridine as the most suitable probe structure. The utility of the dienophile probe was then demonstrated in bacterial extracts. Tylactone, novodaryamide and daryamide A, piperazimycin A, and the saccharamonopyrones A and B were cleanly labeled in extracts from their respective bacterial producers, in high regioselectivity but with varying degrees of diastereoselectivity. Further application of the method led to the discovery of a new natural product called nocarditriene, containing an unprecedented epoxy-2,3,4,5-tetrahydropyridine structure, from marine-derived Nocardiopsis strain CNY-503.


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The potential for the discovery of new biologically-active natural products from microorganisms is still promising, provided new methods to identify and isolate these molecules are invented. The most striking evidence for this view is the disparity between the large number of biosynthetic gene clusters present in bacterial genome sequences and the relatively low number of characterized natural products.1,2 Some of the gene clusters are “silent” and not expressed under standard growth conditions. However, many natural products are simply produced in low amounts and obscured by the presence of other metabolites. Transcriptomic and mass spectrometry-based metabolomic studies of the Salinispora bacteria, for instance, demonstrate that the current characterized set of known secondary metabolites is only the “tip of the iceberg.”3,4 One promising approach to natural product discovery that we have been pursuing complements traditional bioactivity-guided isolation and utilizes the inherent reactivity of a metabolite to inform the detection and isolation process. According to this method, termed “reactivity-guided isolation” (RGI), chemical probes consisting of a UVMS tag and a chemoselective reagent are designed to label natural products containing a specific pharmacophore or functional group. The probe structure and reaction conditions must be finely tuned so that the probe reacts exclusively with the desired functionality in a mixture of sugars, lipids, peptides, and various secondary metabolites that constitute a crude extract. To date, reactivity-guided methods have been restricted to simple functional groups that are ubiquitous in primary metabolism,5–8 with very few exceptions including our thiol-based method for the identification of electrophilic natural products.9–11 Methods that, instead, target structural units common to secondary



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metabolism are inherently more valuable for the discovery of new secondary metabolites. Polyketides are formed from a series of Claisen condensation reactions between thioester-bound subunits that are catalyzed by acyl transferase (AT), ketosynthase (KS) and acyl carrier protein (ACP) domains. Ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains partially or completely reduce the polyketide. Successive modules with a KS-AT-DH-KR-ACP domain architecture produce conjugated dienes (n = 2), trienes (n = 3), tetraenes (n = 4), etc., where n is the number of modules. Thus, a conjugated alkene correlates well with polyketide metabolism. Although this functionality does not represent a pharmacophore per se, many natural products with a conjugated alkene nonetheless possess remarkable biological properties. For example, spiramycin (1) is a diene-containing antibiotic and antiparasitic drug, and bufalin (2) is a pyronecontaining cardiotonic steroid isolated from toad venom (Figure 1). Rapamycin (3) is an immunosuppressant drug used to prevent organ rejection.12,13 A semisynthetic derivative of rifamycin, rifampicin (4) is an antibiotic used for the treatment of several types of bacterial infection, including tuberculosis,14 and avermectin B1a (5) is an insecticide whose significance was underscored by Omura’s 2015 Nobel Prize in Medicine.15 As expected, the biosynthetic gene clusters of the microbial metabolites 1 and 3-5 have been demonstrated to contain the KS-AT-DH-KR-ACP motif, within the srmG gene (modules 2-3) for spiramycin,16 the rapB gene (modules 8-10) for rapamycin,17 the rifE gene (modules 9-10) for rifamycin,18 and the aveA3 gene (modules 9-10) for avermectin.19


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The nitroso Diels-Alder (NDA) reaction is a well-established, concerted, chemoand regioselective cycloaddition that may well be exploited as a general method for the discovery

of

conjugate

alkene-containing

polyketides

like

rapamycin

and

avermectin.20,21 NDA reactions with nitrosobenzenes have been known for a century.22 In 2007, Miller and co-workers described the synthesis and reactivity of nitrosopyridines in NDA reactions.23–25 Furthermore, the application of these reagents for the derivatization of complex diene-containing natural products such as leucomycin A7, ergosterol, and colchicine was also detailed.



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Figure 1. Conjugated alkene-containing compounds spiramycin (1), bufalin (2), rapamycin (3), rifampicin (4), avermectin B1a (5).


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Results We thought to exploit the NDA reaction, particularly using bench-stable nitrosoarenes, for the discovery of conjugated alkene-containing natural products in unprocessed extracts. We installed a bromine atom onto each of our candidate nitrosoarene “probes” since this atom gives a unique signature in a mass spectrometer due to the 1:1 abundance of two bromine isotopes (79Br:81Br). 4-Bromonitrosobenzene (6) was prepared in one step from 4-bromoaniline using oxone (Figure 2).26 5-Bromo-2nitrosopyridine (7) and isomers 8-10 were prepared in a two-step oxidation from the corresponding aminobromopyridines.23,27

Figure 2. The nitroso Diels-Alder reaction, bromonitrosobenzene 6 and bromonitrosopyridines 7-10.



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We first evaluated the five candidate probes using spiramycin (1) as substrate (Figure 3). Diene 1 exhibits a nondistinct UV/vis absorption profile with a single λmax = 232 nm under typical LC-MS conditions (acetonitrile, water, 0.1% formic acid). Reaction of spiramycin with nitrosobenzene probe 6 required prolonged heating at 60 ºC in tetrahydrofuran (THF) to yield products 11a/11b as a mixture of diastereomers in 64% yield. In contrast, reaction of spiramycin with nitrosopyridine probe 7 was complete within 3 h at room temperature, again yielding a mixture of diastereomers 12a/12b in 59% yield. The superior reactivity of nitrosopyridines in NDA reactions is welldocumented.25 The major diastereomers 11a and 12a were purified and characterized by comprehensive 2D NMR analysis. The C-10/C-13 carbon chemical shift [for 12a, Osubstituted: δC = 75.9; N-substituted: δC = 51.0] defined the regioselectivity of the reaction. Although cycloaddition of spiramycin with 8 and 9 also proceeded at room temperature, reaction did not occur between spiramycin and probe 10. The lack of reactivity in 10 is likely due to the proximity of the large bromine substituent to the reactive site. At this point, we decided to proceed with 5-bromonitrosopyridine probe 7 for the remainder of the study since 1) it exhibited favorable reactivity at room temperature compared to 6 and 2) it yielded adducts with prominent UV/vis absorption characteristics compared to those from 8 and 9, including long-wavelength λmax at 260 and 310 nm. We also included 4-bromonitrosobenzene 6 in subsequent reactions, whenever possible, as a benchmark. Lastly, N,N'-bis(5-bromophenyl)diazene N-oxide and N,N'-bis(5-bromopyridin-2-yl)diazene N-oxide, known azoxy by-products of the reaction, were observed in the spiramycin reaction and in subsequent reaction mixtures.27


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Both probes labeled bufalin (2) (see Figure 3). Bufalin was converted to 1,5dihydro-2H-pyrrol-2-ones 13 and 14 using probes 6 and 7, respectively, both at room temperature and in a completely diastereoselective manner. In both cases, the first cycloaddition was followed by decarboxylation and reaction with a second equivalent of probe. Elevated temperatures were not required for the reaction of bufalin and 6 since the natural product is locked in the s-cis conformation. However, the reaction did not proceed to completion at room temperature, and the yield of 13 was therefore low (29%).

Heating led to the formation of several by-products. The mechanism and

structure elucidation of 13 and 14 are discussed in greater detail below. Both probes 6 (24 h at 60 °C) and 7 (3 h at room temperature) likewise labeled rapamycin (3) (see Figure 3). 1H NMR spectra of the reaction mixtures both showed an apparent 1.3:1 mixture of diastereomeric adducts 15a/15b and 16a/16b. The C-1/C-4 carbon chemical shift (for 16a, N-substituted: δC = 57.9; O-substituted: δC = 70.7) defined the regioselectivity of the reaction. The diastereomers could not be separated by reversed-phase HPLC using a variety of stationary phases. This result was unexpected since rapamycin is reported, in a study to make nonimmunosuppresive rapamycin analogs, to react with nitrosobenzene with high diastereoselectivity.28,29 Indeed, repeating the published reaction with nitrosobenzene under the reported conditions also gave a diastereomeric mixture (see Supporting Information). We next examined the reactivity of the probes with more challenging dienecontaining substrates rifampicin (4) and avermectin B1a (5) (see Figure 3). Here, the dienes are 1,1-disubstituted, and so nitroso Diels-Alder reaction demands the formation of a crowded quaternary carbon stereocenter. Although conversion to brominated



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adducts using probe 6 was not observed, treatment of rifampicin (4) with probe 7 at room temperature afforded 17 as a single diastereomer, which was purified in 25% yield and characterized by 2D NMR analysis. The C-16/C-19 carbon chemical shift (Nsubstituted: δC = 67.0; O-substituted: δC = 77.4) defined the regioselectivity of the reaction. Unexpectedly, the first equivalent of probe 7 was consumed in an oxidationreduction reaction to give rifampicin quinone. To the best of our knowledge, rifampicin derivatives resembling 17 have not been reported. Avermectin B1a (5) was not labeled to a significant extent with either probe. Thus, there are certain steric or conformational factors that can prevent efficient labeling of a diene-containing natural product, despite the presence of the KS-AT-DH-KR-ACP sequence tag in the sequenced Streptomyces avermitilis genome.19


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Figure 3. Reaction of nitrosobenzene 6 (THF, 60 ºC, 12 h) and nitrosopyridine 7 (THF, rt, 3 h) with conjugated alkene-containing compounds 1-4. Reaction with bufalin (2) and 6 was conducted at room temperature. Key carbon chemical shifts for products from reaction with 7 are shown. UV/vis profiles of the substrates (1-4), the nitrosobenzene



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adducts (11a, 13, 15a), and the nitrosopyridine adducts (12a, 14, 16a, 17) are depicted for comparison.

We then attempted to understand the reactivity of the nitrosoarenes with bufalin (2). The fact that 2 reacted with two equivalents of probe was clear from LC-MS analysis of the reaction mixture, which showed a dibrominated 1:2:1 isotopic cluster for the product. However, the structure of the adducts 13 and 14 could not be determined with certainty using NMR techniques. Thus, given that the adducts generally exhibit superior crystallinity compared to the parent compounds, we set out to determine the structures by X-ray crystallography. A concentrated solution of 1,5-dihydro-2H-pyrrol-2one 13 in DMSO yielded X-ray quality crystals in short order using the vapor diffusion method. Analysis of the crystal derived in this way revealed 5-imino-dihydro-2H-pyrrol2-one 18 (Figure 4a) (CCDC 1546784). Apparently, a water molecule was lost during the recrystallization attempts, as indicated by an increase in retention time, an 18 amu decrease in molecular mass, and a change in UV/vis profile involving a new long wavelength-absorbing chromophore. Ultimately, we synthesized a model 5-hydroxyamino-1,5-dihydro-2H-pyrrol-2-one by reacting probe 6 with α-pyrone (19) to give 20 in good yield, and this compound gave X-ray quality crystals via slow evaporation from CDCl3 (Figure 4b) (CCDC 1841575). Some conversion of 20 to the corresponding 5-imino-1,5-dihydro-2H-pyrrol-2-one was observed during silica gel chromatography, which decreased the yield of 20. Finally, good agreement between 13/14 and 20 in 1H and 2D NMR data allowed unambiguous assignment of the structure of 13 and 14 (Figure 4c). Eventually, we realized that the


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reaction between α-pyrone and nitrosobenzene to give the corresponding 1,5-dihydro2H-pyrrol-2-one was first reported in 1976.30 Also described was the tendency of 5hydroxyamino-1,5-dihydro-2H-pyrrol-2-ones to dehydrate to the analogous imino compounds, under conditions that include silica gel chromatography. The proposed mechanism for conversion of bufalin to 13/14 is outlined in Figure 4d. Bicyclic intermediate 21 decarboxylates to 1,2-oxazine 22 following the first cycloaddition reaction. Claisen-type rearrangement gives 23. The aldehyde and imine react intramolecularly to give 24, which re-aromatizes to 25. A 1,5-dihydro-2H-pyrrol-2one tautomer, hydroxypyrrole 25 reacts with electrophiles at the 2-position. As nitroso compounds are electrophilic at the nitrogen center, 25 undergoes a rapid addition reaction to give 13/14.

Figure 4. The bufalin reaction. A) 5-Hydroxyamino-1,5-dihydro-2H-pyrrol-2-one 13 dehydrated to 5-imino-1,5-dihydro-2H-pyrrol-2-one 18, whose structure was verified by X-ray crystallographic analysis. B) Using model pyrone 19, the 5-hydroxyamino-1,5dihydro-2H-pyrrol-2-one 20 was isolated from the reaction mixture, and this compound 13 ACS Paragon Plus Environment


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was also subjected to crystallographic analysis. C) HMBC NMR correlations for 13. D) Proposed mechanism for the formation of 13/14 from bufalin. Using 30 mL cultures we then screened ten marine-derived bacterial extracts including species from Streptomyces, Salinispora, Nocardia, Micromonospora, Kocuria, Saccharomonospora, Frankia, and Nocardiopsis using probe 7 at room temperature. The presence of one or more UV-active (254 nm) peaks with a brominated isotope pattern in the LC-MS chromatogram of the reaction mixture indicated a “hit.” Inspection of the LC-MS chromatogram of the original extract was used to exclude endogenous brominated compounds. The UV/vis profiles for the peaks of interest were also examined in order to determine if they conformed to the profiles of “typical” adducts like 11a and 16a. Although the small number of samples allowed for manual inspection of LC-MS data, we applied the recently-developed bioinformatics tool MeHaloCoA to detect brominated cycloadducts in an automated fashion.31 Treatment of extract from Salinispora pacifica strain CNT-084 with probe 7 gave a single brominated compound 26, of unknown structure, with the molecular formula C28H41BrN2O6 [m/z (M+H)+ 581.2384, calcd for C28H42BrN2O6 581.2226] (Figure 5). Although this adduct did not survive isolation and characterization, its formula was used to calculate the molecular formula of the parent natural product (C23H38O5). In addition, analysis of the genome of strain CNT-084 showed that it contained a PKS gene cluster with high sequence homology to the rosamicin gene cluster.2,32 Taken together, we surmised that we had labeled tylactone (27), a diene-containing biosynthetic precursor to rosamicin, which we subsequently purified and characterized.33,34


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When extract from marine Streptomyces sp. CNQ-085 was treated with probe 7, two labeled compounds were produced (see Figure 5). The molecular formula of bromide 28 was determined as C23H29BrN4O6 [m/z (M+Na)+ 559.1160, calcd for C23H29BrN4O6Na 559.1168]. The formula of the parent natural product was then calculated to be C18H26N2O5, corresponding to daryamide A (30), a known weakly cytotoxic natural product from this strain.35 The molecular formula of bromide 29 was determined as C23H27BrN4O6 [m/z (M+H)+ 535.1176, calcd for C23H28BrN4O6 535.1192]. As before, the formula of the parent natural product was then calculated to be C18H24N2O5 [m/z (M+Na)+ 371.1576, calcd for C18H24N2O5Na 371.1583], suggesting that the metabolite is an oxidized analogue of daryamide A. The planar structure of 29 was elucidated following extensive 1D and 2D NMR (COSY, HSQC, HMBC) experiments. Retrosynthetic logic and subsequent characterization of the parent natural product revealed a new daryamide, named novodaryamide (31). COSY and HMBC correlations analogous to 30 defined the propanamide-substituted epoxy-cyclohexenone ring; COSY correlations also defined the reactive (2E,4E)-7-methylocta-2,4-dienamide. Novodaryamide contains the intact epoxyquinone pharmacophore found in manumycin and related metabolites.36 Recently, Fu, et al., synthesized 31 via oxidation of daryamide D.37 Notably, the absolute stereochemistry of the daryamides in the original publication was misassigned because the Mosher’s analysis was interpreted incorrectly.35



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Figure 5. RGI of conjugated alkene-containing natural products in extracts. LC chromatograms at 254 nm are shown both before (black) and after (red) the labeling 16 ACS Paragon Plus Environment

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reaction. A) Treatment of extract from Salinispora strain CNT-084 with 7 yielded brominated cycloadduct 26, which is derived from tylactone (27). B) Extract from Streptomyces strain CNQ-085 gave 28 and 29 after reaction, which are derived from daryamide A (30) and novodaryamide (31). 1H-1H COSY and HMBC correlations are shown for the novodaryamide cycloadduct (29) and novodaryamide (31). “Oxide” refers to N,N'-bis(5-bromopyridin-2-yl)diazene N-oxide.

Four chloro-brominated products in approximately equal amounts were observed when extract from Streptomyces sp. strain CNQ-593 was treated with probe 7 (Figure 6). The molecular formula of C36H50BrClN10O11 was determined from the dominant HRMS ions [m/z (M+Na)+ 935.2418, calcd 935.2430], and the calculated formula (C31H47ClN8O10) matched the chlorinated natural product piperazimycin A (34).31 HPLC purification and subsequent NMR analysis of all adducts confirmed the formation of dihydro-1,2-oxazine products via a Diels-Alder reaction between nitroso probe 7 and the conjugated diene in piperazimycin A. It was apparent that the four products are regioand stereoisomers, suggesting that the diene side chain of 34 is highly flexible and electronically symmetrical. Pyrones 37 [m/z (M+H)+ 239.1639, calcd for C14H23O3 239.1640] and 38 [m/z (M+H)+ 239.1640, calcd for C14H23O3 239.1640] with the molecular formula C14H22O3 were discovered in extracts from Saccharomonospora sp. strain CNQ-490 following a telling reaction of the extract with 7, which produced two brominated compounds, 35 [m/z (M+H)+ 425.0831, calcd for C16H26BrN2O4 425.1076] and 36 [m/z (M+H)+ 425.0835, calcd for C16H26BrN2O4 425.1076], having the molecular formula



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C19H25BrN2O4 (see Figure 6). The amine nitrogen atom of the bromopyridine probe was attached to C-3 on the basis of chemical shift (δC 88.0), but no site of attachment for the oxygen atom was available. We therefore proposed that a hydroxylamino group was present at C-3 in 35 and 36. The parent natural products were then isolated and characterized by NMR. Shared HMBC correlations for 37 from H3-14 (δH 1.95) and H315 (δH 1.94) to C-2 (δC 164.5), C-3 (δC 97.9), C-4 (δC 164.5), C-5 (δC 105.6), and C6 (δC 159.6) defined a 4-hydroxy-substituted α-pyrone. In the same way, HMBC correlations and chemical shift values for 38 also outlined a 4-hydroxy-substituted α-pyrone. A 5methylhexyl substituent was attached to C-6 in 37, whilst a heptane substituent was attached to C-6 in 38. These natural products, called the saccharomonopyrones A (37) and B (38) were very recently described by Nam and Fenical.32 The natural products may react with probe 7 via an addition reaction at the nucleophilic C-3 carbon atom in lieu of an NDA reaction, similar to the reactivity of bufalin intermediate 25 described above (see Figure 4c).22


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Figure 6. RGI of conjugated alkene-containing natural products in extracts. LC chromatograms at 254 nm are shown both before (black) and after (red) the labeling reaction. A) Extract from Streptomyces strain CNQ-593 gave 32a/32b and regioisomers 33a/33b after reaction, which are derived from piperazimycin A (34). B) Extract from Saccharomonospora strain CNQ-490 yielded 35 and 36, which are derived from saccharomonopyrones A (37) and B (38). 35’ and 36’ are isomers of 35 and 36, presumably corresponding to the initial cycloadducts formed upon reaction with 7. 1H-1H COSY and HMBC correlations are shown for both regioisomers of the piperazimycin cycloadduct (32a/33a) and for the saccharomonopyrone A cycloadduct (35) and saccharomonapyrone A (37). “Oxide” refers to N,N'-bis(5-bromopyridin-2-yl)diazene Noxide.

Lastly, treatment of extract from Nocardiopsis sp. strain CNY-503 with probe 7 also showed formation of two brominated products (39a/39b) with the molecular formula C22H28BrN3O3 [m/z (M+H)+ 462.1374 and 462.1375, calcd for C22H29BrN3O3 462.1392] (Figure 7). The natural product (40) in the untreated bacterial extract was targeted for purification by HPLC on the basis of its calculated mass C17H25NO2 [m/z (M+H)+ 276.1957, calcd for C17H26NO2 276.1964]. The chiral compound, [α]D = +30 (c 0.2, DMSO), was isolated as a yellow solid. Its 1H NMR spectrum showed five distinct olefinic protons at δH 6.35 (H-4), 6.05 (H-7), 5.95 (H-5), 5.75 (H-3), and 5.58 (H-8). Two COSY spin systems containing these protons, H3-1 to H-5 and H-7 to H2-9, were connected via HMBC correlations from a methyl group at H3-16 (δH 1.75) to C-5 (δC 130.2), C-6 (δC 130.2), and C-7 (δC 130.2). This 6-methyl-3,5,7-nonatriene moiety


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represents the reactive part of the molecule. A 2D NOESY experiment established the all-E configuration of the triene unit, showing correlations between H-2 and H-4, H-3 and H-5, H-4 and H-16, H-7 and H-9, and H-8 and H-16. An additional COSY spin system extended from methine protons at H-12 (δH 3.70/δC 42.6) to H-13 (δH 3.64/δC 52.9), constituting a cis epoxide, and from H-13 to N-substituted methylene protons at H2-15 (δH 3.24,3.60/δC 42.7). HMBC correlations extending from both ends of this fourcarbon spin system to quaternary carbon C-11 (δC 171.7) indicated a six-membered imine-containing heterocycle fused to the epoxide. Additional HMBC correlations from the methyl group at H3-17 to C-9 (δC 43.4), C-10 (δC 75.0), and C-11 tied the alkyl side chain to the heterocyclic bicycle. The carbon chemical shift at C-10 indicated a tertiary alcohol was present at this position. This new natural product was named nocarditriene. The reactivity of nocarditriene (40) supported our initial structure assignment. When the compound was concentrated from acidified solvents, the chemical shift of C11 shifted considerably downfield to δC 190.7 due to the formation of iminium ion 41. Furthermore, the imine in 40 was hydrolyzed in the presence of acetic anhydride and triethylamine, via the N-acyl iminium species, to form N-acetylated epoxyketone 42 [m/z (M+Na)+ 358.1999, calcd for C19H29NO4Na 358.1994], which was also characterized by NMR. The carbon chemical shift of the newly-formed ketone at C11 (δC 209.1) was evident. Nocarditriene (40) is a polyketide alkaloid containing a rare epoxy-2,3,4,5tetrahydropyridine structure. Unless fused to another carbocycle, as in latumcidin (43), epoxy-tetrahydropyridines have not been observed in other natural products to date.38 Reduction of nocarditriene to the corresponding epoxy-piperidine, which has been



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observed in some cultivations of CNY-503 (unpublished results), revealed the biosynthetic and structural similarities between 40 and cyclizidine (44), indolizomycin (45), streptazone E (46), and several other related metabolites.39–42 As such, the metabolite is likely biosynthesized by a type I PKS, reductively off-loaded and cyclized to the imine.43,44

Figure 7. RGI of the nocarditriene (40). LC chromatograms at 254 nm are shown both before (black) and after (red) the labeling reaction. A) Treatment of extract from 22 ACS Paragon Plus Environment

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Nocardiopsis strain CNY-503 with 7 yielded brominated cycloadducts 39a/39b, as a mixture of isomers, which are derived from nocarditriene (40). The relative configuration of the epoxide moiety is shown. Concentration from acidic solutions (0.1% TFA) yielded iminium ion 41, which is characterized by a C-11 chemical shift of δC190.7. Treatment with acetic anhydride and triethylamine gave ketone 42, which is characterized by a C11 chemical shift of δC 209.1.

Figure 8. Structures of latumcidin (43), cyclizidine (44), indolizomycin (45), and streptazone E (46).

Conclusion Reactivity-guided isolation (RGI) is used to select for specific metabolites in an extract by targeting naturally-occurring structural features. The derivative formed is a highly detectable, often crystalline entity worthy of characterization because its structure reveals the structure of the original natural product following simple retrosynthetic analysis. The subsequent identification of the unlabeled metabolite in the LC-MS chromatogram of unreacted extract is trivial. Provided the appropriate bioassay is in place, these natural products could be isolated using traditional bioactivity-guided isolation (BGI). However, RGI is chemocentric while BGI is completely disconnected 23 ACS Paragon Plus Environment


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from structure. Here, we show that RGI can also complement genome-mining analyses since the latter produce predictions of natural product structure that ultimately need to be validated. When targeting pharmacophores, the labeled metabolite is not expected to retain the biological activity associated with the unlabeled metabolite since the pharmacophore is disrupted in the process.10 But the targeted functional group need not be a pharmacophore. If the metabolite exhibits bioactivity, the non-pharmacophore nature of the functional group, a conjugated alkene in the present case, means that the labeled metabolites may retain this activity. In many cases, however, alteration of the overall 3D structure of the molecule disrupts binding to its original target. In fact, the association of rapamycin with mTOR was deliberately prevented by reacting the natural product with nitrosobenzene.28 The effect on biological activity of nitrosobenzene and nitrosopyridine derivatization for the 11 natural products described above, which yielded 16 isolable derivatives, has not been thoroughly assessed. RGI allows natural products chemists to uncover metabolites with a specified functional group or structural feature that may otherwise be challenging to find in a complex extract. It does not address other issues in the field that thwart discovery efforts, such as the existence of “silent” biosynthetic gene clusters. Certainly, a somewhat exhaustive chemical analysis of any of the extracts mentioned above would lead to the isolation of the very same metabolites, especially those in high abundance, such as nocarditriene. We contend, however, that with automated LC-MS tools like MeHaloCoA and DeltaMS, RGI can be successfully applied to a large number of


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extracts and is capable of identifying inconspicuous metabolites that are in very low abundance and hidden in the muck.31,45



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METHODS Preparation, detection, and purification of 1-bromo-4-nitrosobenzene (6). Nitrosobenzene 6 was prepared according to the literature.46,47 To a solution of 4bromoaniline (2.50 g, 14.5 mmol) in DCM (50 mL) was added a solution of oxone (17.9 g, 29.0 mmol) in water (150 mL). The mixture was stirred vigorously overnight, and the layers were then separated. The aqueous layer was extracted with DCM (50 mL). The combined organic layers were washed with 1 M HCl (30 mL), a saturated NaHCO3 solution (30 mL), water (30 mL), brine (20 mL), dried over Na2SO4, filtered, and concentrated. The product was dry-loaded onto silica gel and purified by silica gel chromatography [80 g silica gel cartridge, 60 mL min‒1, 100% hexanes for 10 min, 0-5% EtOAc in hexanes over 15 min, tR = 12-25 min] to afford, after trituration with methanol to remove color, 1.18 g (44%) of 6 as a white solid. UV/Vis: λmax = 228, 293, 318 nm; 1H NMR (500 MHz, CDCl3): δ 7.78 (m, 4H); 13C NMR (125 MHz, CDCl3): δ 164.0, 132.8, 131.9, 122.3; the exact mass could not be obtained using ESI-MS.

Preparation, detection, and purification of 5-bromo-2-nitrosopyridine probe (7). Nitrosopyridine 7 was prepared in a similar manner to the literature with crucial modifications as described in Howard, et al.23,27 To a solution of 2-amino-5bromopyridine (4.78 g, 27.6 mmol) and dimethylsulfide (2.2 mL, 30 mmol) in DCM (25 mL) at 0 °C was added a solution of N-chlorosuccinimide (3.69 g, 27.6 mmol) in DCM (100 mL) dropwise via cannula over 1 h. After an additional 1 h at 0 °C, the solution was allowed to warm to rt for 1 h. A solution of sodium ethoxide (15 g, 21 wt%, 46 mmol) was then added, followed 10 min later by the addition of water (25 mL). After 4 h at rt,


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additional water (100 mL) was added, and the layers were separated. The aqueous layer was extracted with DCM (50 mL). The combined organic extracts were washed with water, dried over Na2SO4, filtered, and concentrated. To a stirring solution of the mCPBA (13.6 g, 70%, 55.2 mmol) in DCM (200 mL) at 0 °C was added a solution of the crude sulfilimine in DCM (50 mL) dropwise via cannula over 15 min. After 30 min at 0 °C, dimethylsulfide (1.0 mL, 13 mmol) was added. A saturated solution of Na2CO3 (100 mL) was added, and the layers were separated. The organic layer was washed with water, dried over Na2SO4, filtered, and concentrated. The product was dry-loaded onto silica gel and purified by silica gel chromatography [80 g silica gel cartridge, 60 mL min‒ 1

, 0-20% EtOAc in hexanes over 25 min, 20% EtOAc in hexanes for 10 min, tR = 15-33

min] to afford 1.19 g (23%) of 7 as a yellow solid. UV/Vis: λmax = 228, 304, 318 nm; 1H NMR (500 MHz, CDCl3): δ 8.88 (d, J = 2.1 Hz, 1H), 8.20 (dd, J = 8.4, 2.1 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), dimer: δ 8.11 (br s, 1H), 8.07 (dd, J = 8.6, 2.1 Hz, 1H), 7.78 (d, J = 8.6 Hz, 1H); 13C NMR (125 MHz, CDCl3): δ 166.9, 154.1, 150.6, 148.1, 142.4, 141.9, 128.0, 122.8, 120.0, 111.5; HRESI-Q-TOF-MS: m/z (M+Na)+ 208.9326 calcd for C5H379BrN2ONa, found 208.9326. Nitrosopyridines 8-10 were prepared in a similar manner.

Generation of crude extracts. All strains were obtained from SIO actinomycete culture collection Streptomyces sp. CNQ-085, Salinispora pacifica CNT-084, Saccharomonospora sp. CNQ-490, and Nocardiopsis sp. CNY-503 were obtained from and grown in A1 seawater medium (10 g L‒1 of starch, 4 g L‒1 of yeast extract and 2 g L‒1 of peptone in 75% seawater and 25% deionized water) and Streptomyces sp. CNQ-



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593 was grown in a seawater marine medium (chitosan 2 g L‒1, krill powder 2 g L‒1, menhaden meal 2 g L‒1, fish solubles 4 g L‒1, starch 5 g L‒1). From cryovials, these strains were first grown in starter cultures (50 mL) and after 4-5 days the whole volume was used to inoculate culture media (1 L). If necessary, after 3-4 days, 25 mL aliquots of the pre-cultures were used to inoculate producing cultures (1 L). All cultures were incubated at 27 oC with rotatory shaking at 200 rpm for 2-7 days, after which Amberlite XAD-16N and XAD-7HP (~10 g L‒1 each) was added and left shaking for 2 h. The resin was filtered through cheesecloth, washed with deionized water, and extracted with acetone. The acetone was removed under reduced pressure, and the resulting aqueous layer was extracted with ethyl acetate. The combined organic extracts were dried over sodium sulfate, and concentrated.

Labeling reactions. To pure natural product or crude extract, probe 6 or 7 was added dry THF and the reaction vessel sealed. In general, reactions with 6 were heated in an oil bath at 60 oC for 12 h, while reactions with 7 were left stirring at room temperature for 3 h. The progress of the reactions was monitored on an analytical Agilent 1100 Series HP system (1.0 mL min‒1) with UV (210, 254, and 360 nm) and ELS detection and also on an analytical Agilent 1260 Infinity Series LC system coupled to a 6530 Series Q-TOF mass spectrometer, both using a C18(2) Phenomenex Luna column (5 µm, 100 mm x 4.6 mm) with a 10 or 20 min solvent gradient from 10% to 100% acetonitrile + 0.1% formic acid in water. Probe adducts were purified by isocratic reversed-phase HPLC (acetonitrile and water + 0.1% trifluoroacetic acid (if needed)) using a C8(2), C18(2) or CN Phenomenex Luna columns (5 µm, 250 mm x 10 mm) with UV detection.


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ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: XXXXXXX. Detailed experimental procedures, chromatograms of reaction mixtures, UV/vis and MS spectra, and tabulated NMR spectroscopic data including original 1H, 1H-1H COSY, HSQC, and HMBC spectra for all new compounds (PDF). Crystal structure data were deposited at the Cambridge Crystallographic Data Centre as CCDC 1546784 (18) and CCDC1841575 (20) (CIF).

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS This work was supported by start-up funds from the Scripps Institution of Oceanography. G.C.F. is a Howard Hughes Medical Institute Gilliam Fellow. We are grateful to A. L. Rheingold and C. E. Moore from the UCSD Crystallographic Facility for X-ray crystal structure analysis and to Y. Su from the UCSD Molecular Mass



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Spectrometry Facility for high-resolution mass spectrometry measurements. A highresolution LC-MS instrument was provided by the National Institutes of Health (S10 OD0106400).


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